Ovarian carcinoma is a leading cause of cancer death worldwide among women. The molecular mechanisms of ovarian carcinogenesis are not known, although a role for heredity has been suggested. We have shown recently that defects in the mitotic checkpoint are common in ovarian cancer. The mitotic checkpoint is a conserved surveillance system that prevents anaphase onset until all chromosomes are aligned. Loss of mitotic checkpoint results in genomic instability, which is a hallmark of cancer. However, it is still not fully understood how proteins interact to enforce the mitotic checkpoint. The proposed studies are designed to investigate the molecular basis of mitotic checkpoint in mammalian cells and its relevance to genomic instability in ovarian cancer. Specifically, we will: (a) fully characterize newly identified isoforms of mitotic checkpoint proteins MAD1 and CDH1; (b) study the transcriptional regulation of the expression of checkpoint proteins MAD1 and MAD2; (c) characterize the interaction of MAD1-MAD2 with the nuclear pore complex; and (d) identify the upstream signals regulating MAD2B-CDHI. Our studies will shed light on the integration of mitotic checkpoint functions with the programs of cell proliferation and cell death. In all studies the relevance to ovarian cancer will be assessed. In particular, we will: (a) screen ovarian cancer cells and tissues for loss of heterozygosity and mutations in mitotic checkpoint loci; (b) compare the expression and localization patterns of various mitotic checkpoint proteins in ovarian cancer cells and correlate them with checkpoint stringency and oncogenic potential; (c) investigate the genetic and epigenetic causes of mitotic checkpoint defects in ovarian cancer; and (d) study the anti-proliferative mechanisms of mitotic checkpoint-targeting drugs such as taxol and vincristine using ovarian cancer as a model. We have shown in various systems that MAD2 and other checkpoint proteins sensitize cells to mitotic checkpoint-targeting chemotherapeutic drugs. Further elucidation of the causative roles of checkpoint defects for drug resistance will reveal novel strategies for combination therapy.